Method and kit for detecting and verifying copy number variation of different regions of genome

Abstract

The invention discloses a method and a kit for detecting and verifying copy number variation of different regions of a genome. The method comprises the following steps that at least two specific primer pairs are designed for different regions of a genome, the different regions of the genome comprise a single exon and a chromosome segment, and different primer pairs in the designed specific primer pairs are located at different positions of the regions; a to-be-detected genome is taken as a template, the primer pair is adopted for fluorescent quantitative PCR reaction, and copy number variation of the to-be-detected genome is detected and verified according to a quantitative result. The method not only can be used for detection and verification of large-fragment CNV, but also can be used for detection and verification of the copy number of a single exon, and can be used for determining the copy number of genes involved in a CNV overlapping region. The detection result is subjected to multi-level correlation verification, so that the accuracy of the detection result is ensured, the false positive / false negative rate is reduced, the detection period is short, and the cost is low.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and specifically designs a method and a kit for detecting and verifying copy number variations in different regions of a genome. Background Art

[0002] Genetic variations can be divided into single nucleotide variations, small insertions and deletions, and structural variations larger than 50 bp according to their lengths. Deletions and duplications are called copy number variations (CNVs).

[0003] Copy number variation (CNV) is caused by genomic rearrangements and accounts for an order of magnitude greater genomic variation than single-nucleotide polymorphisms (SNPs). Although most of these variations are phenotypic, some have been shown to be associated with a variety of human diseases, the most notable of which are neurodevelopmental disorders, including autism spectrum disorder, schizophrenia, intellectual disability, attention deficit hyperactivity disorder, developmental delay, and epilepsy.

[0004] For many years, chromosome karyotype analysis technology has been considered the "gold standard" for diagnosing chromosomal aberrations, but its detection cycle is long and the resolution is low, and it cannot detect CNVs below 5Mb; although FISH technology can detect chromosomal abnormalities in smaller fragments, it requires the design of specific DNA probes for each different site, which prolongs the detection cycle and increases the cost; comparative genomic hybridization (array-CGH) and single nucleotide polymorphism microarray (SNP-array) are both high-throughput, rapid, automated, independent of cell culture, and applicable to a wide range of sample tissue types. Both can perform fluorescence signal scanning at the whole genome level, and can realize copy number variation detection of the entire chromosome set, detect all chromosome numbers and chromosomal imbalance variations, especially for detecting pathogenic deletions and duplications of small chromosome fragments. Genomic copy number variations (pCNVs) have outstanding advantages, but generally speaking, the resolution of array-CGH is about 6kb and the resolution of SNP-array is about 3kb, and both cannot detect smaller variations; although MLPA testing can detect the copy number of a single exon, the detection results of single exon deletions still need to be verified by other methods; CNV-seq technology developed based on the principles of second-generation sequencing methodology detects genomic CNVs through low-magnification whole-genome sequencing, but the resolution is about 100kb. It is often used to detect large-fragment CNVs at the whole-genome level. Although it can indicate CNVs less than 100kb or smaller, it cannot determine whether they are true or false. Moreover, for CNV boundary regions involving dosage-sensitive genes, other methods need to be used for verification.

[0005] Therefore, based on current technology, a new detection method is urgently needed to solve the problem of insufficient resolution of comparative genomic hybridization (array-CGH) technology, single nucleotide polymorphism microarray (SNP-array) technology and CNV-seq. While supplementing the results or suspected results with verification, single exon copy number detection can also be performed, and the accuracy of the test and verification results can be ensured, the limitations of the technology itself can be overcome, the false positive / false negative rate of the test can be reduced, and the influence of high GC content can be effectively overcome. Summary of the Invention

[0006] Based on this, it is necessary to provide a method and kit for detecting and verifying copy number variations in different regions of the genome.

[0007] The first aspect of the present application provides a method for detecting and verifying copy number variations in different regions of a genome, comprising the following steps:

[0008] Designing at least two pairs of specific primers for different regions of the genome, wherein the different regions of the genome include single exons and chromosome segments, and different primer pairs in the designed specific primer pairs are located at different positions in the regions;

[0009] The genome to be tested is used as a template and the primer pair is used to perform a fluorescent quantitative PCR reaction, and the copy number variation of the genome to be tested is detected and verified based on the quantitative results.

[0010] In some embodiments, for a single exon of a genome, different primer pairs in the designed specific primer pairs have different amplicon lengths and the different primer pairs do not share the same upstream or downstream;

[0011] Optionally, the amplicons of the different primer pairs are located within the entire sequence of a single exon of the genome to be tested.

[0012] In some embodiments, for a chromosome segment of a genome, different primer pairs in the designed specific primer pairs are located at the top, middle, and bottom of the chromosome segment, respectively.

[0013] In some embodiments, when the GC content of different regions of the genome is greater than or equal to 70%, the Tm of the specific primer pair is 68°C~72°C; when the GC content of different regions of the genome is less than 70%, the Tm of the specific primer pair is 55°C~61°C.

[0014] In some embodiments, when the GC content of different regions of the genome is greater than or equal to 70%, the reaction procedure of fluorescent quantitative PCR is 90°C to 95°C for 5 minutes; 95°C for 15 seconds, 65°C for 20 seconds; 34 to 36 cycles;

[0015] When the GC content of different regions of the genome is less than 70%, the reaction procedure of fluorescent quantitative PCR is 90°C~95°C, 3 minutes; 95°C 15 seconds, 60°C 20 seconds; 34~36 cycles.

[0016] In some embodiments, when the GC content of different regions of the genome is greater than or equal to 70%, the initial reaction system of the fluorescent quantitative PCR reaction further includes at least one of betaine and dimethyl sulfoxide;

[0017] Optionally, the amount of betaine solution corresponding to 25 μL of the initial reaction system is 4 μL to 6 μL, the concentration of betaine in the betaine solution is 3 M to 5 M, and the volume of dimethyl sulfoxide is 0.5 μL to 2 μL.

[0018] In some embodiments, according to 2 -ΔΔCt The quantitative results of the values ​​were used to detect and verify the copy number variation of the genome to be tested.

[0019] In some embodiments, the detection validation criteria are: when 2 of the different primer pairs -ΔΔCt When the values ​​are within the same threshold range and the normal controls have no copy number abnormalities, copy number variation exists.

[0020] In some embodiments, when 2 -ΔΔCt When the value is 0, the copy number of the genome to be tested is 0;

[0021] When 2 -ΔΔCt When the value is 0.3-0.7, the copy number of the genome to be tested is 1;

[0022] When 2 -ΔΔCt When the value is 0.8-1.2, the copy number of the genome to be tested is 2;

[0023] When 2 -ΔΔCt When the value is 1.3-1.7, the copy number of the genome to be tested is 3;

[0024] When 2 -ΔΔCt When the value is 1.8-2.2, the copy number of the genome to be tested is 4.

[0025] In some embodiments, the length of the specific primer pair is 80 bp to 700 bp.

[0026] The second aspect of the present application provides a kit for detecting and verifying copy number variations in different regions of the genome, wherein the kit includes the specific primer pairs in the method for detecting and verifying copy number variations in different regions of the genome described in the first aspect of the present application.

[0027] The method for detecting and verifying the copy number variation of different regions of a genome provided in the foregoing can be used for detecting and verifying large fragment CNVs, and can also be used for detecting and verifying the copy number of a single exon, and can determine the copy number of a gene involved in a CNV overlap region. Further, the method can overcome the problem of false negative results caused by a single exon tandem repeat or repeat insertion into other chromosomal regions, and can effectively overcome the influence of high GC content, and can perform multi-level related verification of detection results, ensure the accuracy of detection results, reduce the false positive / false negative rate, and has a short detection period and low cost. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application, and more completely understand the present application and its beneficial effects, the drawings needed in the description of the embodiments or examples will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0029] Figure 1 qPCR amplification curves of primer pair 1, primer pair 2, primer pair 3 and primer pair 4 in step a) of an embodiment of the present application, wherein A is the qPCR amplification curve of primer pair 1, B is the qPCR amplification curve of primer pair 2, C is the qPCR amplification curve of primer pair 3, and D is the qPCR amplification curve of primer pair 4;

[0030] Figure 2 qPCR melting curves of primer pair 1, primer pair 2, primer pair 3 and primer pair 4 in step a) of an embodiment of the present application, wherein A is the qPCR melting curve of primer pair 1, B is the qPCR melting curve of primer pair 2, C is the qPCR melting curve of primer pair 3, and D is the qPCR melting curve of primer pair 4;

[0031] Figure 3 qPCR amplification curves of primer pair 1 in step a) of an embodiment of the present application, wherein A is the qPCR amplification curve of adding 4 μL betaine, B is the qPCR amplification curve of adding 5 μL betaine, and C is the qPCR amplification curve of adding 6 μL betaine;

[0032] Figure 4 qPCR melting curves of primer pair 1 in step a) of an embodiment of the present application, wherein A is the qPCR melting curve of adding 4 μL betaine, B is the qPCR melting curve of adding 5 μL betaine, and C is the qPCR melting curve of adding 6 μL betaine;

[0033] Figure 5qPCR amplification curve for primer pair 4 in step a) of one embodiment of the present application, wherein A is the qPCR amplification curve with 0.5 μΐ of added dimethyl sulfoxide, B is the qPCR amplification curve with 1 μΐ of added dimethyl sulfoxide, and C is the qPCR amplification curve with 2 μΐ of added dimethyl sulfoxide;

[0034] Figure 6 qPCR melting curve for primer pair 4 in step a) of one embodiment of the present application, wherein A is the qPCR melting curve with 0.5 μΐ of added dimethyl sulfoxide, B is the qPCR melting curve with 1 μΐ of added dimethyl sulfoxide, and C is the qPCR melting curve with 2 μΐ of added dimethyl sulfoxide;

[0035] Figure 7 qPCR amplification curve for primer pair 1 in step b) of one embodiment of the present application, wherein A is the qPCR amplification curve with 4 μΐ of added betaine, B is the qPCR amplification curve with 5 μΐ of added betaine, and C is the qPCR amplification curve with 6 μΐ of added betaine;

[0036] Figure 8 qPCR amplification curve for primer pair 2 in step b) of one embodiment of the present application, wherein A is the qPCR amplification curve with 4 μΐ of added betaine, B is the qPCR amplification curve with 5 μΐ of added betaine, and C is the qPCR amplification curve with 6 μΐ of added betaine;

[0037] Figure 9 qPCR amplification curve for primer pair 3 in step b) of one embodiment of the present application, wherein A is the qPCR amplification curve with 4 μΐ of added betaine, B is the qPCR amplification curve with 5 μΐ of added betaine, and C is the qPCR amplification curve with 6 μΐ of added betaine;

[0038] Figure 10 qPCR amplification curve for primer pair 4 in step b) of one embodiment of the present application, wherein A is the qPCR amplification curve with 4 μΐ of added betaine, B is the qPCR amplification curve with 5 μΐ of added betaine, and C is the qPCR amplification curve with 6 μΐ of added betaine;

[0039] Figure 11 qPCR amplification curve for primer pair 4 in step b) of one embodiment of the present application, wherein A is the qPCR amplification curve with 0.5 μΐ of added dimethyl sulfoxide, B is the qPCR amplification curve with 1 μΐ of added dimethyl sulfoxide, and C is the qPCR amplification curve with 2 μΐ of added dimethyl sulfoxide;

[0040] Figure 12qPCR melting curves of primer pair 1 in step b) in one embodiment of the present application, wherein A is the qPCR melting curve after adding 0.5 μL of dimethyl sulfoxide, B is the qPCR melting curve after adding 1 μL of dimethyl sulfoxide, and C is the qPCR melting curve after adding 2 μL of dimethyl sulfoxide;

[0041] Figure 13 qPCR melting curves of primer pair 2 in step b) in one embodiment of the present application, wherein A is the qPCR melting curve after adding 0.5 μL of dimethyl sulfoxide, B is the qPCR melting curve after adding 1 μL of dimethyl sulfoxide, and C is the qPCR melting curve after adding 2 μL of dimethyl sulfoxide;

[0042] Figure 14 qPCR melting curves of primer pair 3 in step b) in one embodiment of the present application, wherein A is the qPCR melting curve after adding 0.5 μL of dimethyl sulfoxide, B is the qPCR melting curve after adding 1 μL of dimethyl sulfoxide, and C is the qPCR melting curve after adding 2 μL of dimethyl sulfoxide;

[0043] Figure 15 qPCR melting curves of primer pair 4 in step b) in one embodiment of the present application, wherein A is the qPCR melting curve after adding 0.5 μL of dimethyl sulfoxide, B is the qPCR melting curve after adding 1 μL of dimethyl sulfoxide, and C is the qPCR melting curve after adding 2 μL of dimethyl sulfoxide;

[0044] Figure 16 qPCR melting curves of primer pair 4 in step b) in one embodiment of the present application, wherein A is the qPCR melting curve after adding 4 μL of betaine, B is the qPCR melting curve after adding 5 μL of betaine, and C is the qPCR melting curve after adding 6 μL of betaine. DETAILED DESCRIPTION

[0045] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0047] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0048] In this application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute a limitation on the scope of protection of this application.

[0049] As used herein, the terms "having," "containing," "including," and "comprising" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0050] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0051] In this application, when referring to the unit of a data range, if the unit is only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0052] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0053] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0054] In the present application, the terms "first", "second", "third" and the like in the "first aspect", "second aspect", "third aspect" and the like are only for the purpose of description and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third" and the like only serve the purpose of non-exhaustive enumeration description and should be understood as not constituting a closed limitation on the quantity.

[0055] In the present application, "greater than or equal to", "greater than or equal to" and "≥" have the same meaning and can be used interchangeably; "less than or equal to", "less than or equal to" and "≤" have the same meaning and can be used interchangeably; "greater than" can be equivalent to ">"; "less than" can be equivalent to "<". In the present application, unless otherwise specified, "greater than or equal to" and "≥" can be considered to provide two schemes of "greater than" and "equal to". In the present application, unless otherwise specified, "less than or equal to" and "≤" can be considered to provide two schemes of "less than" and "equal to".

[0056] In the present application, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, the distribution of the selected values within the numerical interval is considered to be continuous, and includes both numerical endpoints (i.e. the minimum and maximum values) of the numerical interval, as well as every value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, including both endpoint integers and every integer between the two endpoints, it is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise indicated, numerical ranges disclosed herein are to be understood to include any and all sub-ranges subsumed therein. The "values" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" is intended to broadly include types of numerical intervals such as percentage intervals, ratio intervals, value intervals, etc.

[0057] In the present application, the term "Tm" refers to the temperature at which 50% of the double-stranded nucleic acid molecules (such as DNA double strands or primer and template DNA double strands) dissociate into single strands under heating conditions.

[0058] Currently, traditional CNV detection methods have some limitations, such as insufficient resolution of array-CGH technology, SNP-array technology and CNV-seq; the results of MLPA detection for single exon deletion still need to be verified by other methods; and the detection cycle of chromosome karyotype analysis technology and FISH technology is long.

[0059] Based on this, the embodiments of the present application at least provide a method for detecting and verifying copy number variation of different regions of a genome, comprising the following steps:

[0060] S100: designing at least two pairs of specific primer pairs for different regions of a genome, wherein the different regions of the genome include a single exon and a chromosome segment, and different primer pairs in the designed specific primer pairs are located at different positions of the regions;

[0061] S200: performing a fluorescent quantitative PCR reaction on a template of a genome to be tested using the specific primer pairs, and detecting and verifying copy number variation of different regions of the genome to be tested according to the quantitative results.

[0062] In some embodiments, in step S100, for a single exon of a genome, the lengths of amplicons of different primer pairs in the designed specific primer pairs are different, and the different primer pairs cannot share the same upstream or the same downstream.

[0063] In some embodiments, in step S100, for a single exon of a genome, if the single exon to be detected and verified is a deletion, the primer amplicon can be located inside the entire sequence of the single exon of the gene to be detected, or the upstream primer or the downstream primer can be located inside the sequence of the single exon of the gene to be detected, or the primer amplicon can contain the entire sequence of the single exon of the gene to be detected; if the single exon to be detected and verified is likely to be a duplication, the primer amplicon is preferably located inside the entire sequence of the single exon of the gene to be detected.

[0064] It should be noted that if different primer pairs share the same upstream or downstream, even if the lengths of the amplicons are inconsistent, there are still inconsistent detection results, which need to be re-detected or even if the detection results are consistent, there is still a risk of false positives. The primer amplicon located inside the entire sequence of the single exon of the gene to be detected can prevent false negative results, especially when the single exon to be detected and verified is likely to be a duplication. If the single exon is not a tandem duplication, the primer amplicon sequence is not located inside the entire sequence of the single exon, and a false negative result is likely to be obtained.

[0065] In some embodiments, in step S100, for a chromosome segment of a genome, the chromosome segment to be detected and verified is divided into an upper, middle and lower chromosome segment, and different primer pairs in the designed specific primer pairs are located at the upper, middle and lower of the chromosome segment, respectively.

[0066] In some embodiments, in step S100, when the GC content of different genomic regions is greater than or equal to 70%, the Tm of the designed specific primer pair is 68° C. to 72° C. Without limitation, when the GC content of different genomic regions is, but is not limited to, 70%, 75%, 80%, 85%, 90%, 95%, or a value or range between any two of the foregoing values, the Tm of the designed specific primer pair may be, but is not limited to, 68° C., 69° C., 70° C., 71° C., 72° C., or a value or range between any two of the foregoing values.

[0067] In some embodiments, in step S100, when the GC content of different genomic regions is less than 70%, the Tm of the specific primer pair is 55° C. to 61° C. Without limitation, when the GC content of different genomic regions is, but not limited to, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or a value or range between any two of the foregoing values, the Tm of the designed specific primer pair may be, but not limited to, 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., or a value or range between any two of the foregoing values.

[0068] In some embodiments, in step S200, when the GC content in different regions of the genome is greater than or equal to 70%, the reaction procedure of fluorescent quantitative PCR is 90°C~95°C, 5 min; 95°C 15s, 65°C 20s; 34~36 cycles; further, when the GC content in different regions of the genome is greater than or equal to 70%, the reaction procedure of fluorescent quantitative PCR is 95°C, 5 min; 95°C 15s, 65°C 20s; 35 cycles.

[0069] In some embodiments, in step S200, when the GC content of the different genomic regions is less than 70%, the reaction procedure for the fluorescent quantitative PCR is 90°C to 95°C for 3 minutes, 95°C for 15 seconds, 60°C for 20 seconds, and 34 to 36 cycles. Furthermore, when the GC content of the different genomic regions is less than 70%, the reaction procedure for the fluorescent quantitative PCR is 95°C for 3 minutes, 95°C for 15 seconds, 60°C for 20 seconds, and 35 cycles.

[0070] In some embodiments, in step S200, when the GC content of different genomic regions is greater than or equal to 70%, the initial reaction system of the fluorescent quantitative PCR reaction further includes at least one of betaine and dimethyl sulfoxide; further, the amount of betaine solution corresponding to 25 μL of the initial reaction system is 4 μL to 6 μL, the concentration of betaine in the betaine solution is 3 M to 5 M, and the volume of dimethyl sulfoxide is 0.5 μL to 2 μL. Without limitation, the amount of betaine solution corresponding to 25 μL of the initial reaction system can be 4 μL, 5 μL, 6 μL, or a value or range between any two of the above values, the concentration of betaine in the betaine solution can be 3 M, 4 M, 5 M, or a value or range between any two of the above values, and the volume of dimethyl sulfoxide can be 0.5 μL, 1 μL, 1.5 μL, 2 μL, or a value or range between any two of the above values.

[0071] In some embodiments, in step S200, according to 2 -ΔΔCt The quantitative results of the values ​​were used to detect and verify the copy number variation of the genome to be tested.

[0072] In some embodiments, the detection validation criteria are: when 2 of the different primer pairs -ΔΔCt When the values ​​are within the same threshold range and the normal controls have no copy number abnormalities, copy number variation exists. -ΔΔCt If the values ​​are within the same threshold range and the normal controls show no copy number abnormalities, the result is credible; for the chromosome segment, the 2 -ΔΔCt The value must be the same as the 2nd value of the primer pair located in the middle segment. -ΔΔCt The values ​​are within the same threshold range or 2 of the three segment primer pairs -ΔΔCt If the values ​​are within the same threshold range and the normal controls show no copy number abnormalities, the results are credible.

[0073] In some embodiments, when 2 -ΔΔCt When the value is 0, the copy number of the genome to be tested is 0;

[0074] When 2 -ΔΔCt When the value is 0.3-0.7, the copy number of the genome to be tested is 1;

[0075] When 2 -ΔΔCt When the value is 0.8-1.2, the copy number of the genome to be tested is 2;

[0076] When 2 -ΔΔCt When the value is 1.3-1.7, the copy number of the genome to be tested is 3;

[0077] When 2 -ΔΔCtWhen the value is 1.8-2.2, the copy number of the genome to be detected is 4.

[0078] In some embodiments, when 0<2 -ΔΔCt When the value is ≤0.2, if the single exon or chromosome segment to be detected is single copy itself (i.e. male sample), in combination with negative quality control (blank control), DNA quality control and sanger sequencing, it is detected whether the genome to be detected is chimeric deletion. It can be understood that qPCR is relative quantification, which cannot detect chimerism. However, in actual detection process, a high proportion of chimeric deletion occurs in male sample X / Y chromosome gene copy number detection, so this method can be used for detection of male samples. When no amplification occurs in the blank control and the results of multiple experiments of multiple primer pairs are consistent, the problems caused by pollution, experimental operation and random error can be excluded; the adjacent region adjacent to the adjacent region to be detected is detected and verified, and if the detection result is 1 copy, the deviation caused by DNA quality can be excluded. And sanger sequencing of multiple primer pairs of the region to be verified is the target fragment, which indicates that there is chimeric deletion.

[0079] In some embodiments, the length of the designed specific primer pair is 80-700 bp. Without limitation, the length of the designed specific primer pair can be, but is not limited to, 80 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, or a value or range between any two of the above values.

[0080] The method for detecting and verifying copy number variation of different regions of the genome provided above can be used not only for detection and verification of large fragment CNV, but also for single exon copy number detection, overcoming the limitations of traditional detection methods; not only can the copy number of the gene involved in the CNV overlapping region be determined, but also the problem of false negative detection results caused by single exon tandem duplication or duplication insertion into other chromosome regions can be overcome.

[0081] In the second aspect of the present application, a kit for detecting and verifying copy number variation of different regions of the genome is provided, which comprises the specific primer pair in the method for detecting and verifying copy number variation of different regions of the genome described above.

[0082] Some embodiments are provided below.

[0083] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods not specified in the following examples are preferably referred to the instructions given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0084] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0085] Unless otherwise specified, the experimental materials used in the following examples are all commercially available products. The PCR enhancer is betaine or DMSO.

[0086] Example 1

[0087] Details of the preparation experiment of this embodiment:

[0088] Sample requirements: peripheral blood

[0089] The specific experimental operation process of this embodiment is as follows:

[0090] Peripheral blood was used for gDNA extraction using nucleic acid extraction or purification reagents [Susu Medical Equipment No. 20210902].

[0091] 1. qPCR Amplification

[0092] 1.1 Non-GC-rich - single exon / chromosome segment

[0093] Using KAPA SYBR ® Use the FAST qPCR Kit (2× Master Mix Universal) to perform qPCR amplification using gDNA as the template. Prepare the amplification reaction mix on ice according to Table 1 below:

[0094] Table 1

[0095]

[0096] Vortex to mix and centrifuge briefly. Place the PCR tube in a quantitative PCR instrument, select SYBR for the fluorescence channel, and set the reaction conditions: 20 μL PCR system, 35 cycles, 95°C for 3 minutes (95°C for 15 seconds, 60°C for 20 seconds), followed by a melt curve stage (no need to modify the program).

[0097] 1.2 High GC-single exon / chromosome segment

[0098] Perform qPCR amplification using gDNA as a template. Prepare the amplification reaction mix on ice according to Table 2 below:

[0099] Table 2

[0100]

[0101] Vortex to mix and centrifuge briefly. Place the PCR tube in a fluorescent quantitative PCR instrument, select SYBR for the fluorescence channel, and set the reaction conditions: 20 μL PCR system, 35 PCR cycles, 95°C for 5 min; (95°C for 15 s; 65°C for 20 s), followed by a Melt Curve Stage (no need to modify the program).

[0102] 2. RQ value calculation

[0103] Using RQ=2^ (-ΔΔCt) =2^[-(Ct target gene of the test sample - Ct internal reference gene of the test sample) - (Ct target gene of the control sample - Ct internal reference gene of the control sample)] to calculate the RQ value. If the RQ value is 0, it means 0 copies; if the RQ value is 0.3-0.7, it means 1 copy; if the RQ value is 0.8-1.2, it means 2 copies; if the RQ value is 1.3-1.7, it means 3 copies; if the RQ value is 1.8-2.2, it means 4 copies.

[0104] 2. This embodiment optimizes the main steps of the detection method and screens the conditions. The specific contents are as follows:

[0105] 1.1 Optimization of primer Tm values ​​in high GC regions

[0106] Due to the high GC content, complex secondary structures are easily formed, making conventional methods difficult to amplify effectively. Therefore, primers with high Tm (68°C-72°C) and high annealing temperature (72°C) were used for amplification. The amplification primers are shown in Table 3 below:

[0107] Table 3

[0108]

[0109] qPCR amplification was performed using the KAPA SYBR® FAST qPCR Kit (2×Master Mix Universal). The amplification results of primer pairs 1 to 4 were as follows: Figure 1 A~D and Figure 2 As shown in A~D.

[0110] 1.2 Optimization of PCR amplification annealing temperature

[0111] from Figure 1 and Figure 2 It can be seen that although PCR amplification can be successfully achieved in the step of optimizing the Tm value of the primers in the high GC region, the amplification efficiency is low and the melting curve peak is incomplete, which does not meet the requirements. Therefore, the amplification system was optimized.

[0112] a) 4 μL, 5 μL, and 6 μL of betaine (4 M) and 0.5 μL, 1 μL, and 2 μL of dimethyl sulfoxide (DMSO) were added to the high GC qPCR amplification system. The PCR reaction program was 95°C for 5 min, followed by 35 cycles of (95°C for 15 s and 72°C for 20 s). Amplification failed for all four primer pairs, with no S-shaped amplification or melting curves. The results for primer pair 1 are shown in Figure 1. Figure 3 and Figure 4 As shown, Figure 3 and Figure 4 A to C in the figure were added with 4 μL, 5 μL and 6 μL of betaine (4 M) respectively. The results of primer pair 4 are as follows Figure 5 and Figure 6 As shown in A~C in the figure, A~C are the addition of 0.5μL, 1μL and 2μL of dimethyl sulfoxide respectively.

[0113] b) 4 μL, 5 μL, and 6 μL of betaine (4 M) and 0.5 μL, 1 μL, and 2 μL of dimethyl sulfoxide (DMSO) were added to the high GC qPCR amplification system. The PCR reaction program was 95°C for 5 min, followed by 35 cycles of (95°C for 15 s and 65°C for 20 s). All four primer pairs successfully amplified. Amplification and melting curves are shown in Figure 2. Specifically, the amplification curves for primer pairs 1 through 4 are shown in Figure 2. Figures 7 to 11 As shown, Figures 7 to 10 A~C in the figure were added with 4μL, 5μL and 6μL of betaine (4M), respectively. Figure 11 A to C in the figure were added with 0.5 μL, 1 μL and 2 μL of dimethyl sulfoxide, respectively. The melting curves of primer pairs 1 to 4 are shown in Figure 2. Figures 12 to 16 As shown, Figures 12 to 15 A~C in the above were added with 0.5μL, 1μL and 2μL of dimethyl sulfoxide, respectively. Figure 16 A to C in the figure represent primer pair 4 with betaine (4 M) added at 4 μL, 5 μL, and 6 μL, respectively.

[0114] The experiment found that with increasing betaine or DMSO concentrations, the Tm value of the amplicon decreased, and the melting curve was complete. Combined with the amplification curves, the addition of betaine (4M, 5μL) or DMSO (1μL) resulted in more stable data and better overall results.

[0115] 3. Result Verification

[0116] 1. A sample was collected in an EDTA anticoagulant blood collection tube. The gDNA was extracted using the nucleic acid extraction or purification reagent

Suzhou Machinery Preparation 20210902

[0117] Table 4 qPCR primer information

[0118]

[0119] Table 5 Detection results RQ value

[0120]

[0121] 2. A sample was collected in an EDTA anticoagulant blood collection tube. The gDNA was extracted using the nucleic acid extraction or purification reagent

Suzhou Machinery Preparation 20210902

[0122] Table 6 qPCR primer information

[0123]

[0124] Table 7 Detection results RQ value

[0125]

[0126] 3. A male sample was recruited and CNV-SEQ analysis revealed del(X)(q21.1).seq[GRCh37 / hg19] (80060001-80180000)×0. This region involves the haploid-dose-sensitive gene BRWD3, which is associated with an XLR genetic disorder. However, this sample did not have any clinical phenotype associated with the BRWD3 gene, so testing was required to verify the authenticity of this deletion. A 1 ml peripheral blood sample was obtained and stored in an EDTA anticoagulant tube. DNA was extracted from the blood sample using nucleic acid extraction or purification reagents [Susu Medical Device No. 20210902]. The experiment was performed using the aforementioned experimental protocol. Detailed results are shown in Tables 8 and 9. The RQ values ​​for primer pairs 1, 2, and 3 were all within the 0.3–0.7 threshold range. The RQ values ​​for primer pairs 4 and 5 were both 0. The normal controls were normal to each other, indicating no copy number abnormalities, and the results are reliable. This indicates that the sample has a deletion in the del(X)(q21.1).seq[GRCh37 / hg19] (80060001-80180000) region, and the actual deletion range is much smaller. Furthermore, no copy number abnormalities were observed in the BRWD3 gene, consistent with the clinical phenotype.

[0127] Table 8 qPCR primer information

[0128]

[0129] Table 9 Test results RQ value

[0130]

[0131] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0132] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A method for detecting and verifying copy number variations in different regions of a genome, characterized in that: The following steps are involved: Designing at least two pairs of specific primers for different regions of the genome, wherein the different regions of the genome include single exons and chromosome segments, and different primer pairs in the designed specific primer pairs are located at different positions in the regions; The genome to be tested is used as a template and the specific primer pair is used to perform a fluorescent quantitative PCR reaction, and the copy number variation of the genome to be tested is detected and verified based on the quantitative results.

2. The method for detecting and verifying copy number variation in different regions of the genome according to claim 1, wherein For a single exon of the genome, the amplicon lengths of different primer pairs in the designed specific primer pairs are different and the different primer pairs do not share the same upstream or downstream; Optionally, the amplicons of the different primer pairs are located within the entire sequence of a single exon of the genome to be tested.

3. The method for detecting and verifying copy number variation in different regions of the genome according to claim 1, wherein For the chromosome segment of the genome, different primer pairs in the designed specific primer pairs are located at the top, middle and bottom of the chromosome segment, respectively.

4. The method for detecting and verifying copy number variation in different regions of the genome according to any one of claims 1 to 3, wherein: When the GC content of different regions of the genome is greater than or equal to 70%, the Tm of the specific primer pair is 68°C~72°C; when the GC content of different regions of the genome is less than 70%, the Tm of the specific primer pair is 55°C~61°C.

5. The method for detecting and verifying copy number variation in different regions of the genome according to claim 4, characterized in that When the GC content of different regions of the genome is greater than or equal to 70%, the reaction procedure of fluorescent quantitative PCR is 90°C-95°C, 5 min; 95°C 15 s, 65°C 20 s; 34-36 cycles; When the GC content of different regions of the genome is less than 70%, the reaction procedure of fluorescent quantitative PCR is 90°C~95°C, 3 minutes; 95°C 15 seconds, 60°C 20 seconds; 34~36 cycles.

6. The method for detecting and verifying copy number variation in different regions of the genome according to claim 5, characterized in that When the GC content of different regions of the genome is greater than or equal to 70%, the initial reaction system of the fluorescent quantitative PCR reaction further includes at least one of betaine and dimethyl sulfoxide; Optionally, the amount of betaine solution corresponding to 25 μL of the initial reaction system is 4 μL to 6 μL, the concentration of betaine in the betaine solution is 3 M to 5 M, and the volume of dimethyl sulfoxide is 0.5 μL to 2 μL.

7. The method for detecting and verifying copy number variation in different regions of the genome according to claim 1, wherein According to 2 -ΔΔCt The quantitative results of the values ​​were used to detect and verify the copy number variation of the genome to be tested.

8. The method for detecting and verifying copy number variation in different regions of the genome according to claim 7, wherein: The test validation criteria are: when 2 of the different primer pairs -ΔΔCt When the values ​​are within the same threshold range and the normal controls show no copy number abnormalities, copy number variation exists; Optionally, When 2 -ΔΔCt When the value is 0, the copy number of the genome to be tested is 0; When 2 -ΔΔCt When the value is 0.3-0.7, the copy number of the genome to be tested is 1; When 2 -ΔΔCt When the value is 0.8-1.2, the copy number of the genome to be tested is 2; When 2 -ΔΔCt When the value is 1.3-1.7, the copy number of the genome to be tested is 3; When 2 -ΔΔCt When the value is 1.8-2.2, the copy number of the genome to be tested is 4.

9. The method for detecting and verifying copy number variation in different regions of a genome according to any one of claims 1 to 8, wherein: The length of the specific primer pair is 80 bp to 700 bp.

10. A kit for detecting and verifying copy number variations in different regions of the genome, characterized in that: The kit comprises the specific primer pairs in the method for detecting and verifying copy number variations in different regions of the genome according to any one of claims 1 to 9.

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